The present methods relate to treatments for the amelioration of conditions associated with potential oxidative damage, hypoxia, reperfusion injury, ischemic events, systemic infections, as well as treatments for improving patient conditions worsened by psychological stresses.
The infiltration of damaged tissue by polymorphonuclear neutrophilic leukocytes (neutrophils) and their subsequent activation is a crucial for defense against microbial threats. Once recruited during acute inflammation, neutrophils produce and release copious amounts of reactive oxygen species (ROS) which target potential bacterial invaders. A failure in sufficient production of ROS leads to infections as observed in chronic granulomatous disease (CGD), a disease prompted by a deficient oxidase system in neutrophils [1]. Conversely, excess ROS production is associated with conditions such as chronic wounds [2] and cardiovascular diseases [3]. However, tissue damage can trigger deleterious responses from host defenses, leading to still further tissue damage. There is a need for methods and compositions to enhance healing of an accidental or surgical wound and/or to reduce deleterious effects of endogenous cells, factors and systems in response either to an event such as an injury, an ischemic event, reperfusion injury or potential reperfusion injury, an infarction, such as a cardiac or cerebral infarction, reperfusion of an organ associated with a transplant or the effects of physical and/or psychological stress(es).
Calprotectin, a heterocomplex formed by S100A8 and S100A9, which are two calcium binding proteins, represents 40% of neutrophil cytosolic proteins (by weight) [4]. High serum levels of calprotectin are associated with an immune deficiency, together with growth retardation and arthritis [5, 6, 7]. Work done by others and confirmed by this laboratory has demonstrated that calprotectin regulates neutrophil migration [8-10]. It is shown that S100A8 and S100A9 repel neutrophils in-vitro and that S100A8 inhibits the recruitment of neutrophils in-vivo.
Neutrophil functions are not restricted to ROS or cytokine production. Neutrophils produce and release several serine proteases [11]. Those proteases directly contribute to neutrophil microbicidal action [12] and affect a broad range of biological processes from coagulation [13] to inflammation [14]. The mechanisms of the observed effect of neutrophil derived serine proteases on inflammation involve the activation or inhibition of protease-activated receptors (PARs), a G-coupled family of receptor comprising 4 members named PAR-1 to PAR-4. PARs are activated after proteolysis of an N-terminal portion of the receptors which result in the unmasking of a tethered ligand (for review see Ossovskaya et al [15]). The regulation of PARs activity is a complex process in which proteases can either activate or inactivate the receptors. Neutrophil derived serine proteases including PR3, cathepsin G and elastase participate in the regulation of PARs activity [16]. For example, Cathepsin G activates PAR-4 [17]. PAR-4 partakes in the regulation of inflammatory processes [18, 19] including the recruitment and activation of neutrophils.
Neutrophils isolated from healthy volunteers spontaneously release proteases such as elastase and ROS such as superoxide anion [20] in-vitro. Spontaneous activation of neutrophil oxidative metabolism in-vitro has been widely documented [21] [22-24]. The production and release of ROS by neutrophils is dependent on the NADPH oxidase system and it can be accelerated by phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC) and directly phosphorylates critical subunits of the NADPH oxidase complex [25]. Reports have indicated that exaggerated or reduced rates of spontaneous neutrophil activation are linked to viral [26] or autoimmune conditions [27], implying a physiological function for spontaneous neutrophil activation.
Psychological stress affects physiological functioning both directly via somatic pathways, and indirectly by triggering maladaptive behaviors. Studies have suggested that psychological factors appear to interfere directly with wound healing and closure (Godbout and Glaser, 2006). Marital stress, was associated with a 40% delay in wound healing and a defective immune response (Glaser et al., 1999). Caregivers of patients with Alzheimer's disease who reported emotional conflicts, compared with age-matched non-caregiver controls, also exhibited delays in cutaneous wound healing (Kiecolt-Glaser et al., 1995). A study of oral wounds in dental students revealed that healing proceeded at a 40% slower average rate in oral wounds placed 3 days before examinations, compared with identical wounds placed in the same students during their vacations (Marucha et al., 1998).
Cutaneous wound healing is a multi-step process prone to hypoxia. Conceptually, wound healing can be divided into three sequential but overlapping phases: inflammation, proliferation and remodeling (Thomas et al., 1995). Oxygen metabolism and redox homeostasis are critical in all phases of the healing process. Initially, wounding damages blood vessels and reduces oxygen availability. The early post-wounding acute inflammation phase is characterized by the rapid recruitment and activation of peripheral neutrophils. During their activation neutrophils consume large amount of oxygen to produce antimicrobial reactive oxygen species which profoundly alter the biology of the inflamed tissue. Then the healing process combines an early reduction in blood supply with a substantial increase in oxygen demand, further contributing to potential wound hypoxia.
In the established mouse SKH-1 restraint stress model, psychological stress induces delays in wound closure from day 1 post-wounding. Wound closure is slowed by approximately 30% in mice subjected to stress (Padgett et al., 1998). Stress also results in elevated cortiscosterone levels in the bloodstream (Padgett et al., 1998). Delay in wound closure is associated with disregulated inflammation and defective bacterial clearance. Oxygen metabolism and hypoxia appear to be involved in impaired healing seen in stressed animals. Significantly higher inducible nitric oxide synthase (iNOS) levels are present in wounds of stressed compared with control mice (Gajendrareddy et al., 2005). Hyperoxia resulting from hyperbaric oxygen therapy (HBOT) returns iNOS expression to control levels, and partially or wholly reverses the impairment of wound closure associated with psychological stress (Gajendrareddy et al., 2005).
S100A8 is an oxidation-sensitive anti-inflammatory protein which combines with S100A9 to form the heterocomplex calprotectin. By weight, calprotectin represents 40% of total protein in the neutrophil cytosolic fraction. S100A8 and S100A9 are oxidation sensitive repellent of neutrophils which also inhibit neutrophil chemotaxis toward bacterial products (i.e. formylated peptides) in-vitro (Sroussi et al., 2006; Sroussi et al., 2007). Ala42S100A8, an oxidation-resistant analog of S100A8 engineered using site-directed mutagenesis, retains its chemo-repulsive activity under oxidative conditions, which would otherwise inhibit the wild type S100A8 protein. In the rat air-pouch model of acute inflammation, ala42S100A8 inhibits the recruitment of neutrophils stimulated by bacterial endotoxins (Sroussi et al., 2006).
Previous work has shown that neutrophil depletion results in an accelerated wound closure (Dovi et al., 2003). While neutrophils play an important function in controlling and eliminating bacterial contamination of the wound, reduced neutrophil recruitment and activation seems to be beneficial for wound closure rates possibly by reducing oxygen demand and wound hypoxia. Accordingly, it is hypothesized that S100A8 protein can ameliorate wound healing in a psychological stressed induced model of impaired wounds. Because of the anti-inflammatory nature of S100A8, a secondary aim of this work was to ascertain that S100A8 would not cause a clinically significant defect in wound bacterial clearance. The effect of wild type and ala42S100A8 on wound closure in stressed and non-stressed animals was tested. It was found that ala42S100A8 introduced locally immediately after wounding ameliorated the delay in wound closure rates caused by restraint stress. This beneficial effect occurred without negatively impacting in a clinically significant manner bacterial clearance in the wounds.
There is a need in the art for methods to counteract the deleterious effects of hypoxia in tissues and in bodily fluids so that patient condition can be improved, despite the negative effects of infarct, ischemia, psychological and/or physical stress.